Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Establishment of a femoral critical-size bone defect model in immunodeficient mice.

BACKGROUND: The development of innovative therapies for bone regeneration requires the use of advanced site-specific bone defect small-animal models. The achievement of proper fixation with a murine model is challenging due to the small dimensions of the murine femur. The aim of this investigation was to find the optimal defect size for a murine critical-size bone defect model using external fixation method. METHODS: An external fixation device was attached to the right femur of 30 mice. Femoral bone defects of 1 mm (n = 10), 2 mm (n = 10), and 3 mm (n = 10) were created. Wounds were closed without any additional treatment. To investigate bone healing during the 12-wk observation period, x-ray analysis, histomorphology, immunohistochemistry, and µCT scans were performed. RESULTS: MicroCT analyses after 12 wk showed that 3/8 1-mm defects, 5/8 2-mm defects, and 8/8 3-mm defects remained as nonunions. The defect volumes were 0.36 ± 0.42 mm³ (1-mm group), 1.40 ± 0.88 mm³ (2-mm group), and 2.88 ± 0.28 mm³ (3-mm group; P < 0.001, between all groups). CONCLUSION: Using external fixation, a defect size of 3 mm is necessary to reliably create a persisting femoral bone defect in nude mice.

Melatonin impairs fracture healing by suppressing RANKL-mediated bone remodeling.

BACKGROUND: Melatonin, the major pineal hormone, is known to regulate distinct physiologic processes. Previous studies have suggested that it supports skeletal growth and bone formation, most probably by inhibiting bone resorption. There is no information, however, whether melatonin affects fracture healing. We therefore studied in a mouse femur fracture model the influence of melatonin on callus formation and biomechanics during fracture healing. METHODS AND MATERIALS: Thirty CD-1 mice received 50 mg/kg body weight melatonin i.p. daily during the entire 2-wk or 5-wk observation period. Controls (n = 30) received equivalent amounts of vehicle. Bone healing was studied by radiological, biomechanical, histomorphometrical, and protein biochemical analyses at 2 and 5 wk after fracture. RESULTS: Biomechanical analysis at 2 wk after fracture healing showed a significantly lower bending stiffness in melatonin-treated animals compared with controls. A slightly higher amount of cartilage tissue and a significantly larger callus size indicated a delayed remodeling process after melatonin treatment. Western blot analysis showed a significantly reduced expression of receptor activator of nuclear factor-κB ligand (RANKL) and collagen I after melatonin treatment. The reduced expression of RANKL was associated with a diminished number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts within the callus of the newly formed bone. CONCLUSIONS: Because bone resorption is an essential requirement for adequate remodeling during fracture healing, we conclude that melatonin impairs fracture healing by suppressing bone resorption through down-regulation of RANKL-mediated osteoclast activation.

A new model to analyze metaphyseal bone healing in mice.

BACKGROUND: Despite the increasing clinical problems with metaphyseal fractures, most experimental studies investigate the healing of diaphyseal fractures. Although the mouse would be the preferable species to study the molecular and genetic aspects of metaphyseal fracture healing, a murine model does not exist yet. Using a special locking plate system, we herein introduce a new model, which allows the analysis of metaphyseal bone healing in mice. METHODS: In 24 CD-1 mice the distal metaphysis of the femur was osteotomized. After stabilization with the locking plate, bone repair was analyzed radiologically, biomechanically, and histologically after 2 (n=12) and 5 wk (n=12). Additionally, the stiffness of the bone-implant construct was tested biomechanically ex vivo. RESULTS: The torsional stiffness of the bone-implant construct was low compared with nonfractured control femora (0.23 ± 0.1 Nmm/°versus 1.78 ± 0.15 Nmm/°, P<0.05). The cause of failure was a pullout of the distal screw. At 2 wk after stabilization, radiological analysis showed that most bones were partly bridged. At 5 wk, all bones showed radiological union. Accordingly, biomechanical analyses revealed a significantly higher torsional stiffness after 5 wk compared with that after 2 wk. Successful healing was indicated by a torsional stiffness of 90% of the contralateral control femora. Histological analyses showed new woven bone bridging the osteotomy without external callus formation and in absence of any cartilaginous tissue, indicating intramembranous healing. CONCLUSION: With the model introduced herein we report, for the first time, successful metaphyseal bone repair in mice. The model may be used to obtain deeper insights into the molecular mechanisms of metaphyseal fracture healing.

The LockingMouseNail--a new implant for standardized stable osteosynthesis in mice.

BACKGROUND: Mouse models are of increasing interest to study cellular and molecular mechanisms during fracture healing. However, unlike in large animals and in humans, stable fixation of fractures has been difficult due to the small size of the mouse. METHODS: Based on µCT-scans of a mouse femur, we developed a new intramedullary implant system comparable to a human locking nail. We analyzed fracture healing with osteotomy gap sizes of 0.00, 0.25, and 2.00 mm, which were stabilized with the LockingMouseNail. RESULTS: Femora with a gap size of 0.00 mm and 0.25 mm showed complete fracture healing after 5 wk. Femora showed a secondary bone healing pattern with induction of a small periosteal callus. In contrast, femora with a gap size of 2.00 mm showed sparse periosteal callus formation and a lack of bone bridging even after 10 wk, indicating atrophic non-union. CONCLUSION: The LockingMouseNail allows standardized fixation of mouse femur fractures and also stabilization of segmental defects. By introducing different gap sizes, the healing process can be influenced, ranging from normal fracture healing to atrophic non-union formation. Therefore, the model may ideally be suited to study molecular mechanisms of normal fracture healing, delayed healing, and non-union formation. It may additionally allow studying biological properties and effectiveness of different bone substitutes in stabilized segmental defects.

A novel MRI compatible mouse fracture model to characterize and monitor bone regeneration and tissue composition.

Over the last years, murine in vivo magnetic resonance imaging (MRI) contributed to a new understanding of tissue composition, regeneration and diseases. Due to artefacts generated by the currently used metal implants, MRI is limited in fracture healing research so far. In this study, we investigated a novel MRI-compatible, ceramic intramedullary fracture implant during bone regeneration in mice. Three-point-bending revealed a higher stiffness of the ceramic material compared to the metal implants. Electron microscopy displayed a rough surface of the ceramic implant that was comparable to standard metal devices and allowed cell attachment and growth of osteoblastic cells. MicroCT-imaging illustrated the development of the callus around the fracture site indicating a regular progressing healing process when using the novel implant. In MRI, different callus tissues and the implant could clearly be distinguished from each other without any artefacts. Monitoring fracture healing using MRI-compatible implants will improve our knowledge of callus tissue regeneration by 3D insights longitudinal in the same living organism, which might also help to reduce the consumption of animals for future fracture healing studies, significantly. Finally, this study may be translated into clinical application to improve our knowledge about human bone regeneration.

Development of a stable closed femoral fracture model in mice.

BACKGROUND: Mice have become of increasing interest as experimental model for fracture studies. Due to their small size, most studies use simple pins for fracture stabilization, although insufficient rigidity of fixation critically affects fracture healing. Herein, we studied whether longitudinal fracture compression by an intramedullary screw represents a standardized, stable osteosynthesis technique in mice, and whether it may accelerate fracture healing. MATERIALS AND METHODS: A micro-screw (MouseScrew) was constructed, allowing closed fracture stabilization without traumatizing surgery. Fracture stabilization was achieved by longitudinal compression, which was confirmed by biomechanical testing of osteotomized cadaver femora. Bone repair was analyzed histomorphometrically at 2 and 5 wk after surgery. RESULTS: Ex vivo analyses showed a significantly increased rotational and axial stiffness after screw stabilization (n = 8 each) compared with stabilization techniques using a conventional pin (n = 8 each) or a locking nail (n = 8 each). In the in vivo setting, 2 wk of screw stabilization (n = 8) demonstrated a significantly decreased fibrous tissue formation and an increased cartilage production compared with fractures stabilized by the locking nail (n = 8). After 5 wk callus consisted exclusively of bone in all animals studied without differences between the two stabilization techniques (n = 8 each). CONCLUSIONS: Because prolonged fibrous tissue formation indicates delayed fracture healing, we conclude that the increased stability of the fracture by the use of our newly developed MouseScrew accelerates initial bone repair. Further, this fracture model may represent an ideal tool to study bone repair in mice under conditions of stable fixation.

Genetic variation in mice affects closed femoral fracture pattern outcomes.

The purpose of this study was to determine whether differences in structural and material properties of bone between different mouse strains influence the fracture patterns produced under experimental fracture conditions. Femurs of C57BL/6 (B6), C3H/HeJ (C3H), and DBA/2 (DBA) strains were evaluated using micro-computed tomography (µCT), measurements derived from radiographic images and mechanical testing to determine differences in the geometry and mechanical properties. A fracture device was used to create femoral fractures on freshly sacrificed animals using a range of kinetic energies (∼20-80mJ) which were classified as transverse, oblique, or comminuted. B6 femurs had the lowest bone volume/total volume (BV/TV) and bone mineral density (BMD), thinnest cortex, and had the most variable fracture patterns, with 77.5% transverse, 15% oblique, and 7.5% comminuted fractures. In contrast, C3H had the highest BV/TV, BMD, and thickest cortices, resulting in 97.5% transverse, 2.5% oblique, and 0% comminuted fractures. DBA had an intermediate BV/TV and thickness of cortices, with BMD similar to C3H, resulting in 92.9% transverse, 7.1% oblique, and 0% comminuted fractures. A binomial logistic regression confirmed that bone morphometry was the single strongest predictor of the resulting fracture pattern. This study demonstrated that the reproducibility of closed transverse femoral fractures was most influenced by the structural and material properties of the bone characteristics in each strain, rather than the kinetic energy or body weight of the mice. This was evidenced through geometric analysis of X-ray and µCT data, and further supported by the bone mineral density measurements from each strain, derived from µCT. Furthermore, this study also demonstrated that the use of lower kinetic energies was more than sufficient to reproducibly create transverse fractures, and to avoid severe tissue trauma. The creation of reproducible fracture patterns is important as this often dictates the outcomes of fracture healing, and those studies that do not control this potential variability could lead to a false interpretation of the results.

Characterization of interfragmentary motion associated with common osteosynthesis devices for rat fracture healing studies.

Rat models are widely used in preclinical studies investigating fracture healing. The interfragmentary movement at a fracture site is critical to the course of healing and therefore demands definition in order to aptly interpret the experimental results. Estimation of this movement requires knowledge of the fixation stiffness and loading. The characteristic loading for the rat femur has been estimated, but the stiffness of fixation used in rat studies has yet to be fully described. This study aimed to determine the 6 degree of freedom stiffness of four commonly used implants, two external fixators (RatExFix and UlmExFix), a locking plate, and a locking intramedullary nail, in all degrees of freedom and estimate the interfragmentary movement under specific physiological loads. The external fixator systems allow the greatest movement. Mounted 45° anterolateral on the femur, the RatExFix allows an average of 0.88 mm of motion in each anatomic direction while the stiffer UlmExFix allows about 0.6 mm of motion. The nail is far stiffer than the other implants investigated while the plate allows movement of an intermediate magnitude. Both the nail and plate demonstrate higher axial than shear stiffness. The relatively large standard deviations in external fixator shear motion imply strong dependence on bone axis alignment across the gap and the precise orientation of the specimen relative to the loading. The smaller standard deviation associated with the nail and plate results from improved alignment and minimization of the influence of rotational positioning of the specimen due to the reduced implant eccentricity relative to the specimen axis. These results show that the interfragmentary movement is complex and varies significantly between fixation devices but establishes a baseline for the evaluation of the results of different studies.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and µCT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

Evaluation of high-resolution In Vivo MRI for longitudinal analysis of endochondral fracture healing in mice.

Mice are extensively used for experimental bone-healing studies. However, there are few established nondestructive in vivo techniques for longitudinal fracture-healing analysis in mice, including in vivo micro-computed tomography (µCT) and radiography. Importantly, none of the established methods can discriminate between non-mineralized fibrous tissue and cartilage in the soft fracture callus. Therefore, the objective was to establish high-resolution in vivo magnetic resonance imaging (MRI) for the longitudinal assessment of soft callus formation during bone healing in mice. C57BL/6J mice received a femur osteotomy stabilized using an external fixator and were randomly assigned to five groups. Group 1 mice were scanned three times longitudinally during fracture healing using an optimized MRI scanning protocol to establish an algorithm to characterize the different fracture-callus tissues. Mice of groups 2-4 were scanned once on day 10, 14 or 21, respectively, euthanized after scanning and their femurs subjected to ex vivo µCT and histomorphometric analysis to compare the data assessed by MRI with µCT and histology. Control group 5 mice were not scanned. After 28 days, mice of groups 1 and 5 were euthanized and the fracture-healing outcome was evaluated by bending-test, µCT and histology to determine whether the repeated anesthesia, handling and the MRI measurements themselves influenced fracture healing. The callus-tissue values determined by MRI were mostly comparable to those obtained by µCT and histomorphometric analysis. However, at time points characterized by small relative bone or cartilage areas, MRI measurements were weakly comparable to histomorphometric data, possibly due to the inferior spatial resolution. Importantly, at the early and intermediate phases of healing, cartilage and fibrous-tissue values obtained by MRI were highly accurate. Furthermore, repeated anesthesia, handling and MRI scans did not impact bone healing. Therefore, we demonstrated the feasibility of high-resolution in vivo MRI for longitudinal assessment of soft callus formation during murine endochondral fracture healing.

Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature.

The bone marrow is a central organ of the immune system, which hosts complex interactions of bone and immune compartments critical for hematopoiesis, immunological memory, and bone regeneration. Although these processes take place over months, most existing imaging techniques allow us to follow snapshots of only a few hours, at subcellular resolution. Here, we develop a microendoscopic multi-photon imaging approach called LIMB (longitudinal intravital imaging of the bone marrow) to analyze cellular dynamics within the deep marrow. The approach consists of a biocompatible plate surgically fixated to the mouse femur containing a gradient refractive index lens. This microendoscope allows highly resolved imaging, repeatedly at the same regions within marrow tissue, over months. LIMB reveals extensive vascular plasticity during bone healing and steady-state homeostasis. To our knowledge, this vascular plasticity is unique among mammalian tissues, and we expect this insight will decisively change our understanding of essential phenomena occurring within the bone marrow.

Biomechanical comparison of pin and tension-band wire fixation with a prototype locking plate fixation in a transverse canine patellar fracture model.

OBJECTIVE: To compare a locking plate (LP) with pin and tension-band wire (pin/TBW) for fixation of mid-patellar transverse fractures. MATERIALS AND METHODS: Cadaveric canine stifle joints from 10 adult mixed breed dogs (23-36 kg) were used. Mid-patellar transverse osteotomies were randomly stabilized (in pairs) with either pin/TBW or a prototype LP. Cyclic loads (1 Hz, 500 cycles) at 100% body weight (90°-135° stifle joint extension), were applied. Survival or failure of constructs was defined as <2 mm fracture gap distraction at 500 cycles, or ≥2 mm fracture gap distraction at the number of cycles sustained, respectively. Number of cycles at failure and distraction gap were compared with a paired Student's t-test, and a survival analysis performed with a Mantel-Cox test. All constructs that survived cyclic testing were tested in single cycle load to failure (1.0 mm/sec; 110° stifle joint extension); yield strength was compared with a Wilcoxon rank sum test. Significance was set at p <0.05. RESULTS: All 10/10 LP and three out of 10 pin/TBW fixations survived cyclic testing. Survival analysis, number of cycles at failure, and distraction gap all were significantly different between the two groups (p = 0.0011, p = 0.0013, and p <0.0001, respectively). Construct yield strength was not significantly different (p = 0.1273). CONCLUSIONS: The failure mode with pin/TBW was consistently similar to failures observed clinically. The LP demonstrated consistent, reliable and stable fixation.

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

Adjustable stiffness, external fixator for the rat femur osteotomy and segmental bone defect models.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

A standardized critical size defect model in normal and osteoporotic rats to evaluate bone tissue engineered constructs.

Tissue engineered constructs should be tested for their efficacy not only in normal but also in osteoporotic bone. The rat is an established animal model for osteoporosis and is used often for bone healing studies. In this study a defined and standardized critical size defect model in the rat suitable for screening new tissue engineered constructs in normal and osteoporotic bone is described and validated. Normal and ovariectomised Wistar rats received a unilateral middiaphyseal 5 mm defect in the femur, which was instrumented with a radiolucent PEEK plate fixed with angular stable titanium screws and left untreated. All animals were euthanized eight weeks after defect surgery and the bone healing was evaluated using radiographs, computed tomography measurements, and histology. The developed fixation system provided good stability, even in osteoporotic bone. The implants and ancillary instruments ensured consistent and facile placement of the PEEK plates. The untreated defects did not heal without intervention making the model a well-defined and standardized critical size defect model highly useful for evaluating tissue engineered solutions in normal and osteoporotic bone.

A novel murine femoral segmental critical-sized defect model stabilized by plate osteosynthesis for bone tissue engineering purposes.

Mouse models are invaluable tools for mechanistic and efficacy studies of the healing process of large bone defects resulting in atrophic nonunions, a severe medical problem and a financial health-care-related burden. Models of atrophic nonunions are usually achieved by providing a highly stable biomechanical environment. For this purpose, external fixators have been investigated, but plate osteosynthesis, despite its high clinical relevance, has not yet been considered in mice. We hereby proposed and investigated the use of an internal osteosynthesis for stabilizing large bone defects. To this aim, a 3.5-mm-long segmental bone defect was induced in the mid-shaft of the femur using a Gigli saw and a jig. Bone fixation was performed using a titanium microlocking plate with four locking screws. The bone defect was either left empty or filled with a syngenic bone graft or filled with a coralline scaffold. Healing was monitored using radiographs. The healing process was further assessed using microcomputed tomography and histology 10 weeks after surgery. With the exception of one mouse that died during the surgical procedure, no complications were observed. A stable and reproducible bone fixation as well as a reproducible fixation of the implanted materials with full weight bearing was obtained in all animals tested. Nonunion was consistently observed in the group in which the defects were left empty. Bone union was obtained with the syngenic bone grafts, providing evidence that, although such defects were of critical size, bone healing was possible when the gold-standard material was used to fill the defect. Although new bone formation was greater in the coralline scaffold group than in the left-empty animal group, it remained limited and localized close to the bony edges, a consequence of the critical size of such bone defect. Our study established a reproducible, clinically relevant, femoral, atrophic nonunion, critical-sized defect, low morbidity mouse model. The present study was successful in designing and testing in a small animal model, a novel surgical method for the assessment of bone repair; this model has the potential to facilitate investigations of the molecular and cellular events involved in bone regeneration in load-bearing, segmental-bone defects.

Plunging when drilling: effect of using blunt drill bits.

OBJECTIVE: Plunging when drilling can be a detrimental factor in patient care. There is, although, a general lack of information regarding the surgeon's performance in this skill. The aim of this study was to determine the effect that using sharp or blunt instruments had on the drill bit's soft tissue penetration, using a simulator. MATERIALS AND METHODS: Surgeons taking part in an International Trauma Course were invited to participate. Two groups were defined: experienced and inexperienced surgeons. Twelve holes were drilled in the following order: 3 holes with a sharp drill bit in normal bone (SNB), 3 holes with a sharp drill bit in osteoporotic bone (SOB), 3 holes with a blunt drill bit in normal bone, and 3 holes with a blunt drill bit in osteoporotic bone. Mean values and Student t tests were used for statistical analysis. RESULTS: Thirty-seven surgeons participated, 20 experienced and 17 inexperienced surgeons. Mean plunging depths for SNB, SOB, blunt drill bit in normal bone, and blunt drill bit in osteoporotic bone were, respectively, 5.1, 5.4, 21.1, and 13.9 mm for experienced surgeons and 7.6, 7.7, 22, and 15.9 mm for inexperienced surgeons. Drilling with SNB and with SOB was statistically different, with inexperienced surgeons plunging 2.5 mm (P = 0.31) and 2.6 mm (P = 0.042) deeper, respectively. There was a difference (P < 0.001) between sharp and blunt drill bits in all drilling conditions for both the groups. CONCLUSIONS: Our study showed a significant difference in plunging depth when sharp or bunt drill bit was being used. Surgeons, regardless of their experience level, penetrate over 20 mm in normal bone and over 10 mm in osteoporotic bone.

Sildenafil accelerates fracture healing in mice.

Sildenafil, a cyclic guanosine monophosphate (cGMP)-dependent phospodiesterase-5 inhibitor, has been shown to be a potent stimulator of angiogenesis through upregulation of pro-angiogenic factors and control of cGMP concentration. Herein, we determined whether sildenafil also influences angiogenic growth factor expression and bone formation during the process of fracture healing. Bone healing was studied in a murine closed femur fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analysis at 2 and 5 weeks after fracture. Thirty mice received 5 mg/kg body weight sildenafil p.o. daily. Controls (n = 30) received equivalent amounts of vehicle. After 2 weeks of fracture healing sildenafil significantly increased osseous fracture bridging, as determined radiologically and histologically. This resulted in an increased biomechanical stiffness compared to controls. A smaller callus area with a slightly reduced amount of cartilaginous tissue indicated an accelerated healing process. After 5 weeks the differences were found blunted, demonstrating successful healing in both groups. Western blot analysis showed a significantly higher expression of the pro-angiogenic and osteogenic cysteine-rich protein (CYR) 61, confirming the increase of bone formation. We show for the first time that sildenafil treatment accelerates fracture healing by enhancing bone formation, most probably by a CYR61-associated pathway.

An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.

In most murine fracture models, the femur is stabilized by an intramedullary implant and heals predominantly through endochondral ossification. The aim of the present study was to establish a mouse model in which fractures heal intra-membraneously. Femur fractures of 16 SKH-mice were stabilized by an internal locking plate. Femur fractures of another 16 animals were stabilized by an intramedullary screw. Bone repair was analyzed by radiographic, biomechanical, and histological methods. At 2 weeks, histological analysis showed a significantly smaller callus diameter and callus area after locking plate fixation. Cartilage formation within the callus could only be observed after screw fixation, but not after fracture stabilization with the locking plate. Radiological and biomechanical analysis after 2 and 5 weeks showed a significantly improved healing and a higher bending stiffness of fractures stabilized by the locking plate. Fractures stabilized by the locking plate healed exclusively by intramembranous ossification, which is most probably a result of the anatomical reduction and stable fixation. The fractures that healed by intramembranous ossification showed an increased stiffness compared to fractures that healed by endochondral ossification. This model may be used to study molecular mechanisms of intramembranous bone healing.